We have used functional genomics (i.e., DNA microarrays with follow-up real time quantitative RT-PCR and in situ hybridization analyses) to examine the bladder responses of adult C57B1/6 female mice during the first steps of infection with virulent (FimH+) and isogenic avimlent (FimH-) UPEC strains. Project 3 will extend these analyses with four specific aims. (1) Further characterize the evolution of the response of the adult female C57B1/6 mouse bladder to infection with two genotyped clinical isolates. Our previous studies focused on the acute phase of infection with the cystitis isolate NU14. We will now use DNA microarrays to profile gene expression in the bladders of adult female C57B1/6 mice from 1.5h to 6 weeks after infection with UTI89 (a virulent strain whose geneme will be sequenced in Project 1), and a genotyped asymptomatic bacteriuria (ASB) strain. The temporal evolution as well as the celtular origins of selected responses will be defined using real time quantitative (q) RT-PCR, laser capture microdissection (LCM), in situ hybridization, and multi-label immunohistochemical analyses. (2) Define the impact of specified UPEC genes on the response of the adult female C57B1/6 mouse bladder to infection. We will examine host responses to isogenic strains of UPEC that have engineered mutations in genes expected to alter various steps in the proposed 6 step pathogenic cascade. These mutants will be generated in Project 1 and will include (el) genes targeted based on results obtained from the UT 189 genome sequence, results obtained from differential genotyping of clinical strains, and results garnered from studies of bacterial gene regulation; (b) genes identified during screens of a random transposon-tagged library and (c) mutants in already suspected virulence factors. (3) Define the response of the adult female C57B!/6 mouse bladder to a Gram-positive uropathogen. Enterococcus spp are an important cause of nosocomial infection. A functional genomics-based comparison of the responses to infection with isogenic wild type and fimH strains of an Enterococcus faecalis cystitis isolate will provide insights about how the bladder responds to attachment of Gram-positive uropathegens (4) Define host genes that regulate responses to infection with isogenic wild type and mutant strains of UPEC and E. faecalis. Toll-like receptor (TLR) pathways are involved in the response to infections by both Gram-negative and Gram-positive bacteria. We will perform a comparative functional genomics analysis of the host response to FimH+ UPEC and FimH + E. faecalis in adult female C57BI/6 mice homozygous for wild type tlr alleles, versus the responses of C57B1/6 mice homozygous for tlr4 or tlr2 nuU alleles. We will also examine the contributions of inducible nitric oxide synthase (iNOS), matrilysin (MMP-7) and A20 by infecting genetically engineered C57B1/6 mice homozygous for null alleles of these genes with wild type and mutant strains of bacteria characterized in aims 2 and 3.